Project summary Epigenetic changes to chromatin are linked to aging and cellular senescence and alterations to chromatin structure that result in aberrant gene expression, particularly the loss of silencing, are postulated to cause age- related deleterious effects. In particular, the maintenance of proper chromatin structure and gene expression at telomeres appears to be critical for their protective function and the prevention of genomic instability. Therefore, comprehensive understanding of the epigenetic factors governing chromatin structure at telomeres is key to illuminating the regulatory mechanisms that direct telomere function and their role in longevity. Here, the role of histone lysine methylation in the regulation of chromatin homeostasis at telomeres will be dissected using the model system Saccharomyces cerevisiae. We recently identified Set5, a novel methyltransferase targeting histone H4 at lysines 5, 8 and 12 in budding yeast. Genetic studies showed that Set5 cooperates with a key regulator of transcriptional activity, the H3 lysine 4 methyltransferase Set1, to regulate the organism's ability to respond to stress and to maintain repressive chromatin states near telomeres. Additional genetic evidence further suggests that Set1 and Set5 may also function in genome stability pathways. Here, we will test the hypothesis that Set1 and Set5 are critical regulators of telomeric chromatin architecture required for heterochromatic gene silencing and genome integrity. Two Aims are described: 1) elucidate the Set5- and Set1-dependent dynamics of telomeric chromatin structure, and, 2) test the hypothesis that Set1 and Set5 are critical regulators of telomere function. In the first Aim, chromatin immunoprecipitation and gene expression analysis will be used to characterize the properties of chromatin structure at telomeres that are dependent on these two methyltransferases. In the second Aim, molecular and genetic approaches will be used to investigate the individual and cooperative roles of these methyltransferases in telomere length maintenance and genome stability. Both Set1 and Set5 are conserved histone lysine methyltransferases, therefore these studies will be broadly applicable and provide significant insight in to the role of the orthologous proteins in humans. Combined, this work will advance our understanding of fundamental mechanisms underlying the maintenance of telomeric chromatin homeostasis, as well as open new avenues for the study of potential therapeutic targets that promote healthy telomere function during aging and prevent age-related pathologies.